Ticks without borders: Microbial communities of immature Neotropical tick species parasitizing migratory landbirds along northern Gulf of Mexico.

The long-distance, seasonal migrations of birds make them an effective ecological bridge for the movement of ticks. The introduction of exotic tick species to new geographical regions can lead to the emergence of novel tick-borne pathogens or the re-emergence of previously eradicated ones. This study assessed the prevalence of exotic tick species parasitizing resident, short-distance, and long-distance songbirds during spring and autumn at stopover sites in the northern Gulf of Mexico using the mitochondrial 12S rDNA gene. Birds were captured for tick collection from six different sites from late August to early November in both 2018 and 2019. The highest number of ticks were collected in the 2019 season. Most ticks were collected off the Yellow-breasted Chat (Icteria virens) and Common Yellowthroat (Geothlypis trichas), and 54% of the total ticks collected were from Grand Chenier, LA. A high throughput 16S ribosomal RNA sequencing approach was followed to characterize the microbial communities and identify pathogenic microbes in all tick samples. Tick microbial communities, diversity, and community structure were determined using quantitative insight into microbial ecology (QIIME). The sparse correlations for compositional data (SparCC) approach was then used to construct microbial network maps and infer microbial correlations. A total of 421 individual ticks in the genera Amblyomma, Haemaphysalis, and Ixodes were recorded from 28 songbird species, of which Amblyomma and Amblyomma longirostre was the most abundant tick genus and species, respectively. Microbial profiles showed that Proteobacteria was the most abundant phylum. The most abundant bacteria include the pathogenic Rickettsia and endosymbiont Francisella, Candidatus Midichloria, and Spiroplasma. BLAST analysis and phylogenetic reconstruction of the Rickettsia sequences revealed the highest similarities to pathogenic spotted and non-spotted fever groups, including R. buchneri, R. conorii, R. prowazekii, R. bellii, R. australis, R. parkeri, R. monacensis, and R. monteiroi. Permutation multivariate analysis of variance revealed that the relative abundance of Francisella and Rickettsia drives microbial patterns across the tick genera. We also observed a higher percentage of positive correlations in microbe-microbe interactions among members of the microbial communities. Network analysis suggested a negative correlation between a) Francisella and Rickettsia and, b) Francisella and Cutibacterium. Lastly, mapping the distributions of bird species parasitized during spring migrations highlighted geographic hotspots where migratory songbirds could disperse ticks and their pathogens at stopover sites or upon arrival to their breeding grounds, the latter showing means dispersal distances from 421-5003 kilometers. These findings strongly highlight the potential role of migratory birds in the epidemiology of tick-borne pathogens.

Identification of microRNAs in the Lyme Disease Vector Ixodes scapularis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in many biological processes, including the immune pathways that control bacterial, parasitic, and viral infections. Pathogens probably modify host miRNAs to facilitate successful infection, so they might be useful targets for vaccination strategies. There are few data on differentially expressed miRNAs in the black-legged tick Ixodes scapularis after infection with Borrelia burgdorferi, the causative agent of Lyme disease in the United States. Small RNA sequencing and qRT-PCR analysis were used to identify and validate differentially expressed I. scapularis salivary miRNAs. Small RNA-seq yielded 133,465,828 (≥18 nucleotides) and 163,852,135 (≥18 nucleotides) small RNA reads from Borrelia-infected and uninfected salivary glands for downstream analysis using the miRDeep2 algorithm. As such, 254 miRNAs were identified across all datasets, 25 of which were high confidence and 51 low confidence known miRNAs. Further, 23 miRNAs were differentially expressed in uninfected and infected salivary glands: 11 were upregulated and 12 were downregulated upon pathogen infection. Gene ontology and network analysis of target genes of differentially expressed miRNAs predicted roles in metabolic, cellular, development, cellular component biogenesis, and biological regulation processes. Several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including sphingolipid metabolism; valine, leucine and isoleucine degradation; lipid transport and metabolism; exosome biogenesis and secretion; and phosphate-containing compound metabolic processes, were predicted as targets of differentially expressed miRNAs. A qRT-PCR assay was utilized to validate the differential expression of miRNAs. This study provides new insights into the miRNAs expressed in I. scapularis salivary glands and paves the way for their functional manipulation to prevent or treat B. burgdorferi infection.

An Exploratory Study on the Microbiome of Northern and Southern Populations of Ixodes scapularis Ticks Predicts Changes and Unique Bacterial Interactions.

The black-legged tick (Ixodes scapularis) is the primary vector of Borrelia burgdorferi, the causative agent of Lyme disease in North America. However, the prevalence of Lyme borreliosis is clustered around the Northern States of the United States of America. This study utilized a metagenomic sequencing approach to compare the microbial communities residing within Ix. scapularis populations from northern and southern geographic locations in the USA. Using a SparCC network construction model, we performed potential interactions between members of the microbial communities from Borrelia burgdorferi-infected tissues of unfed and blood-fed ticks. A significant difference in bacterial composition and diversity was found between northern and southern tick populations. The network analysis predicted a potential antagonistic interaction between endosymbiont Rickettsia buchneri and Borrelia burgdorferi sensu lato. The network analysis, as expected, predicted significant positive and negative microbial interactions in ticks from these geographic regions, with the genus Rickettsia, Francisella, and Borreliella playing an essential role in the identified clusters. Interactions between Rickettsia buchneri and Borrelia burgdorferi sensu lato need more validation and understanding. Understanding the interplay between the microbiome and tick-borne pathogens within tick vectors may pave the way for new strategies to prevent tick-borne infections.

Cationic Glycopolyelectrolytes for RNA Interference in Tick Cells.

The black-legged tick (Ixodes scapularis) is the primary vector for bacteria that cause Lyme disease (Borrelia burgdorferi), where numerous glycosylated tick proteins are involved at the interface of vector-host-pathogen interactions. Reducing the expression of key tick proteins, such as selenoprotein K (SelK), through RNA interference is a promising approach to reduce pathogen transmission, but efficient delivery of nucleic acids to arthropods has proven challenging. While cationic glycopolymers have been used as nonviral gene delivery vehicles in mammalian cells, their use in arthropod or insect gene transfection has not been established. In this study, statistical acrylamide-based cationic glycopolymers with glucose or galactose pendant groups were synthesized by reversible addition-fragmentation chain transfer polymerization, and the effects of the saccharide pendant group and cationic monomer loading on polymer cytotoxicity, RNA complexation, and SelK gene knockdown in ISE6 cells were evaluated. All polymers exhibited low cytotoxicity, yet RNA/copolymer complex cell uptake and gene knockdown were highly dependent on the saccharide structure and the N:P (amino to phosphate groups) ratio.